Cytokines are polypeptide hormones that are produced by a cell and affect the growth or metabolism of that cell or another cell. In multicellular animals, cytokines control cell growth, migration, differentiation, and maturation. Cytokines play a role in both normal development and pathogenesis, including the development of solid tumors.
Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF-xcex1) to 140 kDa (Mullerian-inhibiting substance). They include single polypeptide chains, as well as disulfide-linked homodimers and heterodimers.
Cytokines influence cellular events by binding to cell-surface receptors. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors.
Cell differentiation and maturation are also under control of cytokines. For example, the hematopoietic factors erythropoietin, thrombopoietin, and G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow. Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors.
The role of cytokines in controlling cellular processes makes them likely candidates and targets for therapeutic intervention; indeed, a number of cytokines have been approved for clinical use. Interferon-alpha (IFN-xcex1), for example, is used in the treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi""s sarcoma, condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and Puri, xe2x80x9cCommon and Uncommon Features of Cytokines and Cytokine Receptors: An Overviewxe2x80x9d, in Aggarwal and Puri, eds., Human Cytokines: Their Role in Disease and Therapy, Blackwell Science, Cambridge, Mass., 1995, 3-24). Platelet-derived growth factor (PDGF) has been approved in the United States and other countries for the treatment of dermal ulcers in diabetic patients. The hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683). G-CSF, GM-CSF, IFN-xcex2, IFN-xcex3, and IL-2 have also been approved for use in humans (Aggarwal and Puri, ibid.). Experimental evidence supports additional therapeutic uses of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in injured baboon arteries (Giese et al., Restenosis Summit VIII, Poster Session #23, 1996; U.S. Pat. No. 5,620,687). Vascular endothelial growth factors (VEGFs) have been shown to promote the growth of blood vessels in ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and have been proposed for use as wound-healing agents, for treatment of periodontal disease, for promoting endothelialization in vascular graft surgery, and for promoting collateral circulation following myocardial infarction (WIPO Publication No. WO 95/24473; U.S. Pat. No. 5,219,739). A soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News 16(17):5-6, 1996). Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. J Cancer 76:1410-1415, 1997). More recently, thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al., Baillixc3xa8re""s Clin. Haematol. 11:427-445, 1998).
In view of the proven clinical utility of cytokines, there is a need in the art for additional such molecules for use as both therapeutic agents and research tools and reagents. Cytokines are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media.
It is an object of the present invention to provide novel polypeptides, polynucleotides encoding them, and methods of making them.
It is another object of the invention to provide compositions and methods for modulating the proliferation, differentiation, migration, and metabolism of responsive cell types and for regulating tissue development.
Within one aspect of the invention there is provided an isolated polypeptide comprising at least nine contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4. Within one embodiment, the polypeptide has from 15 to 1500 amino acid residues. Within another embodiment, the polypeptide comprises at least nine contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4 operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:7. Within further embodiments, the polypeptide comprises at least 30 contiguous residues of SEQ ID NO:2 or SEQ ID NO:4. Within other embodiments, the polypeptide comprises residues 41-55 of SEQ ID NO:2, residues 56-77 of SEQ ID NO:2, residues 78-92 of SEQ 1) NO:2, residues 78-92 of SEQ ID NO:4, residues 93-110 of SEQ ID NO:2, residues 111-125 of SEQ ID NO:2, residues 111-125 of SEQ ID NO:4, residues 126-148 of SEQ ID NO:2, residues 126-148 of SEQ ID NO:4, or residues 149-163 of SEQ ID NO:2. Within additional embodiments, the polypeptide comprises residues 41-163 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5; residues 34-163 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5; residues 34-178 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5; or residues 18-178 of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:5.
Within a second aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter, a DNA segment encoding a polypeptide as disclosed above, and a transcription terminator. Within one embodiment, the DNA segment comprises nucleotides 52 to 534 of SEQ ID NO:6. Within another embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
Within a third aspect, the invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the DNA segment. The cell can be used within a method of making a polypeptide, the method comprising culturing the cell under conditions whereby the DNA segment is expressed and the polypeptide is produced, and recovering the polypeptide. Within one embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell and recovered from a medium in which the cell is cultured.
Within a further aspect of the invention there is provided a polypeptide produced by the method disclosed above.
Within another aspect, the invention provides an antibody that specifically binds to the polypeptide disclosed above.
Within an additional aspect, the invention provides a method of detecting, in a test sample, the presence of an antagonist of zalpha33 activity. The method comprises the steps of (a) culturing a cell that is responsive to zalpha33; (b) exposing the cell to a zalpha33 polypeptide in the presence and absence of a test sample; (c) comparing levels of response to the zalpha33 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and (d) determining from the comparison the presence of an antagonist of zalpha33 activity in the test sample.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.